The present invention relates to a compound exhibiting anti-heparin activity, of formula Z-Bm-(AXA)x-Bn-(AXA)y-Bo-(AXAz-Bp, to the diagnostic reagents comprising it and to the use of said compound in an in vitro diagnostic test or for producing a medicinal product for anti-heparin purposes.
Unfractionated heparin (UFH) is a natural sulfated polysaccharide of the glycosaminoglycan family. It is extracted essentially from pig or bovine intestinal mucosa.
It is a product which is heterogeneous by virtue of the composition of its saccharide units; as a result, the ionic charge of the molecule is also heterogeneous: different degree of sulfation of the glucosamine sulfate units and number of carboxylic functions different in the iduronic and glucuronic acid units [1, 2].
The essential physical characteristics of heparin are: its polyanionicity and its high charge density. Specifically, each disaccharide carries 3 to 4 negative charges (sulfates and carboxylates).
A particular arrangement of the various units of heparin forms the pentasaccharide reproducibly throughout the entire length of the sequence. This pentasaccharide is known to be the antithrombin III recognition site of heparin, generating between these two entities a high affinity and, consequently, a high anticoagulant activity [3].
The chains containing no pentasaccharide are virtually devoid of anticoagulant activity (only ⅓ of the heparin molecule exhibits this affinity for ATIII) [4].
Unfractionated heparin (UFH) is a polydisperse macromolecule: commercial UFH-based preparations have a molecular weight of between 12 000 and 18 000 Da.
These preparations are mainly used for the purpose of preventive or curative action against thromboses.
It is possible to fractionate UFH by size (after chemical treatment), which produces the family of LMWHs (low molecular weight heparins) [5].
UFH has equivalent activity on factors IIa and Xa. On the other hand, LMWHs maintain a high anti-Xa activity and an anti-IIa activity which decreases with the size of the LMWH molecule.
Since a high anti-IIa activity may have hemorrhagic side effects, the LMWHs can be used advantageously as anticoagulants in place of UFHs.
However, the use of UFH or LMWH heparins in therapy may require the administration of compounds which neutralize their anticoagulant effect, in particular at the end of interventions on the circulation system, such as extracorporeal circulation for example. Moreover, in certain diagnostic tests in vitro, the presence of heparin in the samples may lead to erroneous interpretations of the results. It therefore proves to be necessary to also have compounds with anti-heparin properties for diagnosis.
Protamine sulfate is the only known compound capable of neutralizing the anticoagulant effects of heparin in vivo. Protamine belongs to the family of arginine residue-rich basic proteins, is purified from salmon sperm [6-7] and neutralizes heparin by virtue of its positive charges [8-10].
However, protamine causes hemodynamic and hematologic side effects such as hypotension, bradycardia, thrombocytopenia and leukopenia [11-15]. Specifically, it has been demonstrated that the positive charges on protamine are directly proportional to the efficacy and neutralization of heparin, but also to the toxicity of the molecule [16].
Poly (L)-lysine and polybrene are other compounds known to neutralize heparin. However, these polycations also appear to be too toxic for clinical use.
Wakefield et al. (WO 95/13083) describes protamine variants which are thought to be less toxic. They are peptides having a 20 to 40 amino acid sequence, with a positive charge of between +14 and +18, and the structure of which corresponds to clusters of positive residues interspersed with neutral amino acids. Harris et al. (EP 999219) describes linear or branched anti-heparin peptides comprising a succession of arginine and alanine clusters.
However, the abovementioned documents do not comprise any teachings regarding the need to have anti-heparin agents in diagnosis.
A subject of the present invention is novel synthetic molecules which are of use as heparin-neutralizing agents.
The properties of the molecules of the present invention have been more particularly studied in reagents of in vitro diagnosis. Besides their ability to be insensitive to the effects of the heparin possibly present in the blood samples tested, it may also be necessary for some of these diagnostic reagents to exhibit low turbidity. This property is particularly important in the case of reagents containing thromboplastin for determining the Quick time, in particular on automated devices with optical detection.
The molecules having general formula I explained below make it possible to satisfy these two criteria. They therefore prove to be of particular use in neutralizing the possible interference associated with the presence of heparin, both for diagnostic and for therapeutic purposes.
Thus, the present invention relates to a compound exhibiting anti-heparin activity, of general formula I below:
Z-Bm-(AXA)x-Bn-(AXA)y-Bo-(AXA)z-Bp
in which
Z has the general formula: 
with Cy=-covalent bond
xe2x80x94CH2xe2x80x94
xe2x80x94COxe2x80x94
xe2x80x94COxe2x80x94Sxe2x80x94
xe2x80x94(CH2)nxe2x80x94Xxe2x80x94
with 1xe2x89xa6nxe2x89xa62 and X=Oxe2x80x94, Sxe2x80x94, 
with Cx=-covalent bond
xe2x80x94(CH2)nxe2x80x94, 1xe2x89xa6nxe2x89xa65
or 
with R1=xe2x80x94OH, xe2x80x94NH2 
B represents a basic amino acid and can advantageously be selected from lysine, arginine, ornithine, histidine, homolysine and homoarginine; m, n, o and p represent, independently of one another, an integer of between 2 and 4.
Among the preferred compounds of the invention, the compounds in which m, n and o are equal to 4 and p is equal to 2 are more particularly noted.
AXA represents a hydrocarbon-based chain corresponding to the formula:
xe2x80x94NHxe2x80x94(CH2)nxe2x80x94COxe2x80x94,
n being an integer of between 2 and 6;
x, y and z represent, independently of one another, an integer of between 1 and 6.
Advantageously, Z is selected from the following compounds:
Preferably, Z is pyroglutamic acid (PGlu).
Among the preferred compounds of the invention, the compounds in which AHA corresponds to NHxe2x80x94(CH2)5xe2x80x94COxe2x80x94, i.e. aminohexanoic acid with x equal to 2, and y and z equal to 1, are more particularly noted.
Advantageously, the invention is directed toward the compound corresponding to the following formula:
PGlu-K4-(AHA)2-K4-AHA-K4-AHA-K2
The compounds described above exhibit an anti-heparin capacity, determined by the xcex94 relative clotting time between a heparinized plasma and a nonheparinized plasma, of less than 10%, preferably less than 5%.
A subject of the present invention is therefore also the use of a compound corresponding to formula 1 indicated above, in a reagent for in vitro diagnosis, in particular reagents for calculating the clotting time of a blood sample, or detecting or assaying various elements of the clotting cascade, independently of the presence or absence of heparin in said sample.
Said reagents, in particular reagents of the PT (prothrombin time) type, advantageously exhibit a turbidity, measured by an OD at 630 nm, of less than 0.5, preferably less than 0.1.
According to another aspect, the invention relates to a reagent for in vitro diagnosis, comprising a compound as defined by general formula 1 above, preferably a compound in which AXA corresponds to NHxe2x80x94(CH2)5xe2x80x94COxe2x80x94, i.e. hexanoic amino acid, with x equal to 2, and y and z equal to 1, and more preferentially the compound below of formula:
PGlu-K4-(AHA)2-K4-AHA-K4-AHA-K2
Such a diagnostic reagent thus makes it possible to carry out tests or assays in blood samples, independently of the presence or absence of heparin in said samples.
A reagent according to the invention advantageously corresponds to conventional clotting tests, i.e. tests for determining the clotting time of a blood sample, or to tests for detecting or assaying various elements of the clotting cascade, such as, for example, assaying factor VIIa, fibrinogen, and proteins C and S, the PT test. These tests are commonly used by those skilled in the art.
A preferred reagent for in vitro diagnosis according to the invention is a reagent comprising thromboplastin, for carrying out a PT (prothrombin time or Quick time) test.
The final concentration in such reagents of the compound according to the invention defined above is advantageously between 2 and 10 xcexcg/ml.
Preferably, this concentration is 5 xcexcg/ml.
The invention also relates to the use of a compound of general formula 1 defined above, as an anti-heparin agent for preparing a medicinal product.